Most tumors are characterized by impairment of p53 pathway, either by mutation of the p53 gene (TP53) (Soussi, T. p53 alterations in human cancer: more questions than answers. Oncogene, 2007, 26:2145-56), or by deregulation of other components of the pathway (Vogelstein, B., Lane, D., Levine, A. J. Surfing the p53 network. Nature, 2000, 408(6810):307-10).
The importance of p53 function as a tumor suppressor is underlined by the fact that at least 50% of human tumors carry mutations in TP53. Interestingly, the majority of the TP53 alterations are missense mutations leading to the expression of full-length point mutants (hereinafter identified also as mut-p53 or mutant p53) that accumulate to high levels in tumor cells and show a prolonged half life compared to wild-type protein p53 (herein after also as wt p53).
The determinant role of p53 as tumor suppressor is related to the fact that p53 is a transcription factor that, in response to stress signals, becomes activated and determines different cellular outcomes, as temporary growth arrest and DNA repair, irreversible growth arrest or apoptosis. Furthermore, p53 activity is tightly regulated by several coordinated mechanisms that ensure proper activation including post-translational modifications, as well as interaction with protein partners that modulate its function (Vogelstein, B. et al. 2000, ref. cit.).
The protein p53 may be divided into three functional domains, an N-terminal transactivation domain (hereinafter identified also as TAD), a central DNA binding core domain (hereinafter identified also as CD), comprised from the amino acids (aa) 94 to 298, and a C-terminal oligomerization domain (hereinafter identified also as OLD). Due to multiple splicing, alternative promoter and alternative initiation of translation p53 is also present in several isoforms (Murray-Zmijewski, F., Lane, D. P. and Bourdon, J. C. p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress. Cell death and differentiation, 2006, 13:962-972). The modular structure of p53 is also shared by the other p53 family members, such as p63 and p73, that are also present within the cells in several isoform (Murray-Zmijewski, F. et al., 2006, ref. cit.). Most frequently, tumor-associated mutations are found in the CD. As a consequence of these mutations, the ability of the protein to recognize p53 responsive elements (RE) on DNA is lost and mutant proteins are defective for wild-type function (Joerger, A. C. & Fersht, A. R. Structure-function-rescue: the diverse nature of common p53 cancer mutants. Oncogene, 2007, 26:2226-42). According on the effect of mutations on protein structure, p53 mutants were classified as contact mutants, when an amino acid directly involved in protein-DNA interaction is mutated, or conformational mutants, when mutations alter protein conformation without affecting amino acids involved in DNA binding.
Basing on the high frequency of mutation and on the observation that p53 point mutants are highly abundant in tumors, it was proposed that mutant proteins may play an active role in tumorigenesis. Indeed, the expression of p53 point mutants was shown to favor tumorigenesis and this oncogenic function has been explained by both trans-dominant suppression of wt p53 activities and by the acquisition of novel properties by mutant proteins, commonly referred to as gain-of-function (GOF) (Sigal, A., and Rotter, V. Oncogenic mutations of the p53 tumor suppressor: the demons of the guardian of the genome. Cancer Res., 2000, 60:6788-6793). This is supported by several experimental findings indicating that p53 point mutants exert distinct tumorigenic activities independently of wt p53 inhibition. Mutant p53 GOF has been associated with enhanced tumorigenic potential in mice, increased proliferation and resistance to drugs commonly used in anti-cancer therapy (Iwakuma, T. and Lozano, G. Crippling p53 activities via knock-in mutations in mouse models. Oncogene, 2007, 26:2177-2184). In addition, mouse models have provided evidence for a role of mutant p53 in altering tumor spectrum and increasing the metastatic potential of tumors cells (Lang, G. A., Iwakuma, T., Suh, Y. A. et al. Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome. Cell, 2004, 119(6):861-72; Olive, K. P., Tuveson, D. A., Ruhe, Z. C., et al. Mutant p53 gain of function in two mouse models of Li-Fraumeni syndrome. Cell, 2004, 119(6):847-60; Hingorani, S. R., Wang, L., Multani, A. S. et al. Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice. Cancer Cell, 2005, 7(5):469-83). Conversely, ablation of mutant p53 expression in human tumor-derived cell lines reduced proliferation, survival, chemoresistance and tumorigenicity (Bossi, G., Lapi, E., Strano, S., Rinaldo, C., Blandino, G., Sacchi, A. Mutant p53 gain of function: reduction of tumor malignancy of human cancer cell lines through abrogation of mutant p53 expression. Oncogene, 2006, 25(2):304-9)
The mechanisms underlying mutant p53 GOF remain largely unclear, nevertheless recent experimental findings have shown that mutant p53 is involved in precise cellular events. Mutant p53 was shown to alter the expression of several genes involved in cell proliferation, most likely by modifying the activities of other transcription factors (Weisz, L., Oren, M., and Rotter, V. Transcription regulation by mutant p53. Oncogene, 2007, 26: 2202-2211).
In addition, p53 mutants form aberrant protein complexes with several proteins such as p73, p63 and Mre11 (Li, Y., and Prives, C. Are interactions with p63 and p73 involved in mutant p53 gain of oncogenic function? Oncogene, 2007, 26:2220-2225.; Song, H., Hollstein, M., Xu, Y. p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM. Nat. Cell Biol., 2007, 9(5):573-80) interfering with their ability to induce apoptosis or a proficient DNA damage response respectively.
Given the active role of mutant p53 in tumorigenesis, it represents an interesting target for the development of anti-cancer therapies. Furthermore, since p53 is often mutated only in tumor cells and not in the adjacent normal tissue, a strategy based on inactivation of its pro-oncogenic function would be highly selective. Indeed, some approaches aimed to restore wild-type function to p53 mutants leaded to the identification of small molecules, such as CP-31398 and PRIMA-1 (Selivanova, G. and Wiman, K. G. Reactivation of mutant p53: molecular mechanisms and therapeutic potential. Oncogene, 2007, 26:2243-2254), able to selectively induce massive apoptosis in cells expressing mutant p53.
More recently in WO 2006/054138 another approach to ablate the oncogenic gain-of-function of mutant p53 is disclosed. The purpose in this case has been pursued through a group of small (8-10 aa) peptides able to break the complexes between the mutant p53 and p63, p73 and the respective isoform proteins, increasing in this way the free p73 and p63 and therefore restoring the oncosuppressor activity of these proteins.
However, given the active role of p53 mutants in promoting tumorigenesis, the need to identify various strategies, being able to inactivate their tumorigenic function or to restore wild-type function of p53, in order to find out new very selective and efficient chemotherapeutic treatments of tumors is still strongly felt.